As reported in W098/08932, protein toxins from the genus Photorhabdus have been shown to have oral toxicity against insects. The toxin complex produced by Photorhabdus luminescens (W-14), for example, has been shown to contain ten to fourteen proteins, and it is known that these are produced by expression of genes from four distinct genomic regions: tca, tcb, tcc, and tcd. WO98/08932 discloses nucleotide sequences for many of the native toxin genes.
Of the separate toxins isolated from Photorhabdus luminescens (W-14), those designated Toxin A and Toxin B have been the subject of focused investigation for their activity against target insect species of interest, for example corn rootworm. Toxin A is comprised of two different subunits. The native gene tcda (SEQ ID NO:1) encodes protoxin TcdA (see SEQ ID NO:1). As determined by mass spectrometry, TcdA is processed by one or more proteases to provide Toxin A. More specifically, TcdA is an approximately 282.9 kDA protein (2516 aa) that is processed to provide TcdAii, an approximately 208.2 kDA (1849 aa) protein encoded by nucleotides 265-5811 of SEQ ID NO:1, and TcdAiii, an approximately 63.5 kDA (579 aa) protein encoded by nucleotides 5812-7551 of SEQ ID NO:1.
WO 01/11029 discloses nucleotide sequences that encode TcdA and TcbA and have base compositions that have been altered from that of the native genes to make them more similar to plant genes. Also disclosed are transgenic plants that express Toxin A and Toxin B.
We have observed that heterologous expression of Toxin A does not afford the level of oral toxicity to insects that is observed for the native toxin. It would be very valuable if means could be found to enhance the level of toxicity of heterologously expressed Toxin A.
The present invention provides nucleotide sequences for two genes, tcdB and tccC2, from the tcd genomic region of Photorhabdus luminescens W-14. These sequences were not previously known. We have discovered that coexpression of tcdB and tccC2 with tcdA in heterologous hosts results in enhanced levels of oral insect toxicity compared to that obtained when tcdA is expressed alone in such heterologous hosts. Coexpression of tcdB and tccC2 with tcdA or tcbA, or with any other functionally equivalent toxin gene in the same family as tcdA and tcbA, to provide enhanced oral insect activity falls within the scope of the invention.
SEQ ID NO: 1 is the DNA sequence for tcdA from Photorhabdus luminescens W-14.
SEQ ID NO: 2 is the amino acid sequence for TcdA from Photorhabdus luminescens W-14.
SEQ ID NO: 3 is the DNA sequence for tcdB from Photorhabdus luminescens W-14.
SEQ ID NO: 4 is the amino acid sequence for TcdB from Photorhabdus luminescens W-14.
SEQ ID NO: 5 is the DNA sequence for tccC2 from Photorhabdus luminescens W-14.
SEQ ID NO: 6 is the amino acid sequence for TccC2 from from Photorhabdus luminescens W-14.
SEQ ID NO: 7 is the DNA sequence for tcbA from from Photorhabdus luminescens W-14.
SEQ ID NO: 8 is the amino acid sequence for TcbA from Photorhabdus luminescens W-14.